11. Stimulated emission depletion microscopy (STED)#
Note
If you want to read up more about STED, I recommend the following reviews:
Vicidomini, G., Bianchini, P. & Diaspro, A. STED super-resolved microscopy. Nat Methods 15, 173–182 (2018).
This chapter on STED partially follows M. Fazel, K. S. Grussmayer, B. Ferdman, A. Radenovic, Y. Shechtman, J. Enderlein, S. Pressé, Fluorescence Microscopy: a statistics-optics perspective, [OA, arXiv:2304.01456].
11.1. STED microscopy#
STED microscopy and its generalization RESOLFT, are based upon a traditional confocal microscope (with confocal pinhole in the detec- tion arm) allowing higher resolution imaging while retain- ing the axial sectioning of confocal microscopy.
The spatial resolution improvement is achieved by adding a second de-excitation (depletion) laser quenching or preventing fluorescence around the excitation point and thus confining fluorescence emission to a sub-diffraction limited spot. Stimulated emission is one means by which to de- populate excited states. In this nonlinear process, theoretically described by Albert Einstein, the incoming photon triggers the molecule in the excited state to decay to its ground state, emitting a photon with a phase, frequency, polarization, and momentum identical to the incident photon.
In STED, the spatial and temporal overlap and patterning of the fluorescence excitation and of the depletion laser are crucial. First, fluorophores are excited by a diffraction-limited Gaussian laser beam. If we wait until molecules spontaneously decay (without stimulated emission), no gain in resolution will be achieved. Therefore, it is necessary to introduce the second step where a fraction of the fluorophores are depleted or switched off before they have a chance to emit a fluorescence photon using a torus, or donut-shaped beam whose central minimum coincides with the Gaussian excitation maximum. The depletion laser shape is of course also diffraction-limited. The recorded signal only originates from the “donut hole” far narrower than the original Gaussian beam waist.
11.2. STED resolution#
STED resolution improvement is direct consequence of the physics, and does not need elaborate postprocessing to achieve super-resolution. The STED spatial resolution can theoretically be arbitrarily small provided high enough depletion intensity (I → ∞). In practice, this is not possible and the resolution is limited by the properties of the fluorophores used (and their absorption cross-section of the depletion beam), uncor- rected aberrations of the STED pattern (no good zero) or misalignment of the STED pattern and the excitation laser, the signal-to-noise ratio as due to depletion much less photons are recorded compared to confocal imaging, as well as the STED beam’s relatively high power and thus propensity for fluorophore photobleaching/photodestruction.